Fibrinogen is an antioxidant that protects beta-lipoproteins at physiological concentrations in a cell free system.

Oxidation of beta-lipoproteins has been linked to the development of arteriosclerosis. Using a copper mediated cell free system to oxidize beta-lipoproteins, we found that beta -lipoproteins isolated from plasma were less susceptible to oxidation than lipoproteins from serum and that this was probably due to inhibition by fibrinogen, because removal of fibrinogen from plasma enhanced oxidation, while addition of fibrinogen restored inhibition. Fibrinogen inhibited conjugated diene formation and peroxide formation assayed by the xylenol orange assay (absorbance+/-confidence interval: 0.155+/-0.007 with fibrinogen vs 0.255+/-0.014 without) and retarded copper mediated oxidation of apolipoproteins in low density lipoproteins, reducing the distance of electrophoretic migration by 5 mm. The effect of fibrinogen was not due to chelation of copper, since it provided protection when hydrogen peroxide was substituted for copper as an oxidizing agent. At normal physiological concentration equivalents, fibrinogen showed superior antioxidant properties compared to albumin, melatonin, vitamin C and vitamin E and was superior to the vitamins when compared on an equimolar basis. Other studies have shown the fibrinogen to be more oxidizable than other major plasma proteins and to inhibit peroxide production. Because of its high mass concentration, we postulate fibrinogen is an important antioxidant protecting beta-lipoproteins in plasma and that it may be important in protecting lipoproteins in tissue spaces.

Rapid solubilization and grouping of antigens from beta-hemolytic streptococci via a centrifugal analyzer.

The precipitin reaction between antigens from groups A, B, C, and G betahemolytic streptococci and group-specific antisera was monitored quantitatively by a turbidimetric assay on a centrifugal analyzer. A grouping procedure is described in which antigens from standardized concentrations of bacteria taken directly from agar plates are extracted by Streptomyces albus enzyme at 52 C during a 20-min incubation and then multiple samples assayed in 4 min. Because all components of the system are quantitatively standardized via a spectrophotometer, subjectivity common to other grouping methods is reduced, and high levels of quality assessment can be ensured by establishing numerical control limits. One hundred isolates were assayed using group-specific antisera for A, B, C, and G (400 samples), and 100% correct positive identification with no false positive was obtained. The assay can also serve as a simple way for commercial sources to provide activity information for their products.

Measurement of circulating Clq precipitable immune complexes by a nephelometric type assay after polyethylene glycol extraction in model system.

Clq precipitins were assayed in a model system by absorption nephelometry. Polyethylene glycol was used to extract immune complexes so that sufficient sensitivity with minimal interference could be achieved. Using a double beam spectrophotometer, we found that the reaction of immune complexes with Clq occurred rapidly and plateaued between 5-20 minutes. The optimal concentration of sodium in the reaction mixture was between 0.155-0.165 M, while chloride and EDTA did not affect the reaction significantly. Standard curves generated from heated aggregated gamma globulin showed a high degree of linearity, between 12.5-400 mg/L. Need for conductivity meters, radioisotopes, and other special equipment was eliminated because highly purified Clq is not required for nephelometric assay, and all necessary parameters can be monitored by flame photometry and electrophoresis, which are available in most clinical laboratories. Day to day reproducibility showed CVs of 5.4, 9.5, and 11.4% at control levels of 210, 160, and 70 mg/ L of immune complexes, respectively. Twelve patients with elevated levels of immune complexes by other methods were differentiated from normals by the present method. Nephelometry may offer a simplified approach for testing immune complexes and may become more widespread in clinical laboratories.

The relative usefulness of automated apolipoprotein A-I and high-density lipoprotein cholesterol assays as markers for coronary artery disease.

Research studies have shown that apolipoprotein A-I (apo A-I) is a better marker for coronary artery disease (CAD) than high-density lipoprotein cholesterol (HDLC). Yet, it is unclear whether assays for apolipoprotein A-I, which is part of macromolecular lipoprotein complex, can perform as well when measured by routine automated clinical laboratory techniques. The purpose of this study was to compare automated apolipoprotein A-I assays with HDLC as a marker for CAD. The authors studied two groups of angiographically documented men, aged 44-70 years, 42 with CAD and 123 without CAD in an unmatched, controlled study. Standard clinical laboratory techniques for assaying HDLC, and automated kit rate immunonephelometric, end point immunonephelometric, and immunoturbidimetric assays for apolipoprotein A-I were used. High-density lipoprotein cholesterol was a better marker than apolipoprotein A-I, according to the Mann Whitney test; HDLC also showed better diagnostic sensitivity, specificity, and predictive value. Using a precipitation method, HDL3 was a better marker than HDL2, but not as good as total HDLC. The authors concluded that HDLC remains the best routine single CAD marker commonly available for evaluation of HDL status in a high-risk population.

Interference with glycated hemoglobin by hemoglobin F may be greater than is generally assumed.

An automated electrophoretic method to measure glycated hemoglobin (Hb A1) was compared with manual affinity methods. Good correlation between methods was found. The electrophoretic method showed good run-to-run precision, good linearity, was free from interference by the labile aldimine fraction, and required less time and considerably less consumable expense than the affinity methods. However, as previously reported, Hb F comigrating with Hb A1 caused spurious increases in glycated Hb levels as compared with the affinity methods. This effect was linearly dependent on the Hb F concentration. Using the discrepancy between concentrations of Hb A1 by electrophoresis and glycated hemoglobin by affinity methods, 330 patients were screened in two hospitals for Hb F. A 12% frequency of elevated Hb F (defined as a level that is more than 2%) was found in patients from a community-tertiary care hospital, which is significantly greater than the 1.5% frequency commonly thought to occur in the adult population at large, whereas patients from a Veterans Administration Hospital showed an elevated frequency of 2.2%. Based on this and other studies, it is concluded that the frequency of elevated Hb F in adults may very substantially among different medical centers. The authors recommend against using this method and suggest that laboratories that persist in using it should periodically assess the frequency in patients with Hb F levels greater than 2% of total hemoglobin, replacing the method if an unacceptably high frequency is found.

The relationship between apolipoprotein B-100 and low-density lipoprotein cholesterol after treatment with an HMG CoA reductase inhibitor.

Measurement of apolipoproteins AI and B-100 has been shown to provide at least equivalent information to measurement of lipoprotein cholesterol levels for evaluating risk of cardiovascular disease. In the present study, the authors examined the relationship between APO B-100 and low-density lipoprotein (LDL) cholesterol in a hypercholesterolemic population that was treated with 16 weeks of hydroxymethylglutaryl CoA reductase inhibitor therapy to lower plasma cholesterol levels. The average decrease in APO B-100 was -0.27 g/L (23% of baseline), which was similar to the decrease in LDL cholesterol, -1.6 mmol/L (-0.61 g/L) (30% of baseline). Correlation data (r = 0.902) indicated that the information provided by the two parameters corresponded in individual cases. Apolipoproteins assayed on automated equipment by kit methods are simpler, more straightforward, and provide more reproducible results than measurement of lipoprotein cholesterols. The authors conclude that, in addition to being more reliable for appraising risk of coronary artery disease, measurement of apolipoproteins may be equally useful for monitoring lipoprotein-lowering therapy.

Antibody multispecificity in immunoassay interference.

Recent findings indicate that many endogenous antibodies exhibit multispecificity. These antibodies exhibit a potential for interference with immunoassays. Antibodies that interfere with immunoassays have been called heterophile or heterophilic antibodies. The purpose of this review is: (1) to identify the nature of heterophile antibodies; (2) to delineate the processes that produce them; (3) to examine the mechanisms by which these antibodies cause interference; and (4) to explore how this information can be used to reduce immunoassay interference. In addition to producing specific antibodies, the process of antibody production gives rise to rudimentary antibodies that are polyspecific; e.g., the antigen-combining site has an affinity for antigens of different chemical composition. This process also generates idiotypic antibodies containing cross-reactive idiotopes. These antibodies along with rheumatoid factors, which are themselves polyspecific and rich in cross-reactive idiotopes, are inherent parts of the process of antibody production, and exhibit multispecificity. Mechanisms by which these antibodies cause immunoassay interference are outlined. These properties of antibodies may have substantial consequence in directing future assays toward greater clinical predictive value.

Anti-IgG type test to assay circulating immune complexes from polyethylene glycol precipitates compared with C1q binding and Raji cell tests.

Immune complexes (IC) from over 100 normal and patient serum samples were assayed by turbidimetry using anti-IgG as the indicator following a three-step extraction with polyethylene glycol. For 70 patient samples, a high linear correlation was found between the present assay and the C1q-binding test (r = 0.812). Correlation with the Raji cell test was poor (r = 0.176). Both the present test and the Raji cell test showed greater sensitivity for identifying sera with elevated IC levels than the C1q binding test. The present approach may be accomplished using indicators other than turbidimetry, such as light scatter and isotopically-labelled anti-IgG.

An algorithmic approach using kappa/lambda ratios to improve the diagnostic accuracy of urine protein electrophoresis and to reduce the volume required for immunoelectrophoresis.

The most sensitive routine method for identifying urinary monoclonal immunoglobulin kappa and lambda light chains, called Bence Jones proteins (BJPs), in clinical laboratories is immunofixation electrophoresis (IFE), but this procedure is time-consuming and expensive. As a result, many laboratories screen for paraproteins with urine protein electrophoresis (UPE), which is insensitive when low concentrations of BJP are present and is difficult to interpret with severe proteinuria. The purpose of this study was to determine whether kappa/lambda ratios can be used in conjunction with UPE to improve diagnostic reliability in identifying paraproteins, and decrease the need for IFE on all samples. Urine specimens from 243 patients were examined by UPE and kappa/lambda ratios, and compared with IFE. Due to poor analytical sensitivity, the urinary kappa or lambda concentrations could not be determined in many cases. As a result, many specimens showed kappa/lambda ratios that were indeterminate. Nevertheless, when both urinary kappa and lambda concentrations were undetectable, a BJP could be ruled out. A urinary kappa/lambda ratio between 0.75-3 also ruled out a BJP. The use of kappa/lambda ratios, in conjunction with UPE, resulted in a 52% decrease in the volume of IFE during the course of this study, with 100% sensitivity for detecting BJP.

Immunonephelometric/turbidimetric apolipoprotein B assays for the clinical laboratory.

Because apolipoproteins are a part of complex macromolecular particles, modifications to the assay system may substantially alter results of immunological measurement. Accuracy as analytical recovery cannot be effectively determined by adding exogenous apolipoproteins because antibody access differs from access to endogenous apolipoproteins. Clinical studies are essential for determining accuracy in terms of clinical effectiveness. Since different kit methods use different reagent systems, the purpose of the present study was to compare total cholesterol and LDL cholesterol as markers for coronary artery disease with apo B by automated rate nephelometric, end-point nephelometric and turbidimetric kit methods. The subjects were age matched, male patients with and without angiographically documented coronary artery disease. High correlation coefficients (0.95-0.96) between the assays for both the normal and disease groups indicate that the methods are providing similar information: apo B was a better marker for coronary artery disease (CAD) than total or LDL cholesterol on the basis of univariate, multivariate and Bayesian statistics and correlated best with non-HDL cholesterol. Apo B along with HDLC could explain the variability between the CAD and normal groups without LDLC, total C, or triglycerides.

Recovery of immune complexes from serum-based samples is dependent on the concentration of reactants: heat-inactivated and unheated samples compared by nephelometric assay.

We examined heat-treated and untreated samples for immune complexes (IC) with a nephelometric type Clq binding assay. Curves of absorbance vs. concentration derived from aggregated gamma-globulin in serum samples showed curvilinear behavior, while curves obtained from aqueous samples were highly linear. Curves from heat-treated sera bent upward, while those from unheated sera tended to level off, causing the curves to cross. Heated sera showed greater recovery of IC in the high range and unheated sera greater recovery of IC in the low. Recovery from serum-based samples varied as the concentrations of both Clq and IC in the reaction mixture changed when aqueous curves were used as calibrators. This behavior in serum-based samples seems to be caused by endogenous binders of IC, and may be an important reason for inter-laboratory variation in results from similar samples.

Immunoglobulins from the sera of immunologically activated persons with pairs of electrophoretic restricted bands show a greater tendency to aggregate than normal.

From in vitro data, it has been speculated that pairs of endogenous restricted bands migrating in close proximity in the gamma region upon high resolution serum electrophoresis (HRE) represent circulating immune complexes (CIC). Using a polyethylene glycol (PEG) method to separate CIC, we found a very high correlation between the presence of such band pairs and elevated levels of CIC (CHI2 = 25.7, p less than 0.001) in 51 sera. HRE appears to be a good screening technique to identify, with a high degree of certainty, samples with elevated levels of CIC for delineation by more specific methods. Yet, examination by gel-filtration chromatography and precipitation with PEG indicated that the molecules comprising the band patterns were not CIC, but polyclonal 7S IgG. These bands are usually found in patients with chronic activation of the immune system. Fractionation of the sera from 5 such patients with various cuts of PEG indicated that the average IgG concentration in the 2.5-5%, and 5-7.5% cuts from patients was 3.99 g/l and 2.2 g/l, while from healthy subjects the concentrations were 0.68 g/l and 2.88 g/l. This reversed precipitation pattern was seen both for absolute levels of IgG and for percent of total IgG. On the average the amount of precipitation of IgG in the 2.5-5% fraction of patients was about 5-fold above that seen in the healthy subjects. The endogenous bands were not associated with any specific cut of PEG, but appeared to be proportionally distributed in accord with the levels of IgG. The data is consistent with the idea that immunologically activated patients exhibit a greater tendency for immunoglobulins to associate than normal. This propensity to aggregate may cause CIC to form in situ in local compartments even though CIC do not appear to be present upon analysis by biochemical techniques.

Relationships between triglycerides, lipoproteins, glucose and coronary artery disease.

We examined the relationship of apolipoprotein B (apo B), glucose, triglycerides and other lipoprotein lipids to coronary artery disease (CAD). Using receiver-operating characteristic curves (ROC), we noticed that the triglyceride ROC curve crossed above other lipoprotein curves at a triglycerides level of approximately 1.4 g/l. We examined subgroups of < 1.4 g/l and > 1.4 g/l. ANOVA (F = 18.9, P < 0.0001) and stepwise logistic regression (P = 0.0002) indicated that triglycerides were the best predictor in the < 1.4 g/l group. The best markers in the > 1.4 g/l group were low density lipoprotein cholesterol and apo B. Glucose did not appear to significantly alter the predictive power of triglycerides. These data suggest that triglyceride appears to be an overall significant univariate marker for CAD because of its effect at lower concentrations. The strong relationship between CAD and triglycerides at low triglyceride levels may reflect increased levels of very low density lipoprotein metabolites in some individuals. We conclude that some triglyceride-rich particles are independently atherogenic, that glucose did not alter this relationship and that when the samples were split into those with high and low levels of triglycerides, triglycerides and apo B but not HDLC was a significant predictor of CAD.

Simpler technique for measuring oxidative susceptibility by heparin affinity column isolation of lipoproteins.

Variations in the in vitro oxidative susceptibility or resistance of lipoproteins have been used to test the effect of ingested antioxidants and may prove to be a marker for coronary artery disease. Here we describe a simple technique for isolating and oxidizing beta-lipoproteins that may have utility in the clinical laboratory. Electrophoretic profiles showed that beta-lipoproteins were separated from alpha-lipoproteins and essentially from most other serum proteins using heparin affinity columns. Lipoproteins were normalized in the reaction mixture by measuring apo B in the beta-lipoprotein eluate using an automated apo B method, which gave good recoveries of 106-112%. Copper mediated oxidation was monitored by measurement of conjugated dienes formation at 234 nm for 300 min. When the reaction mixture included beta-lipoprotein eluate containing 0.03 g/L of apoB and 5 micromol/L copper sulfate, conditions were effective for obtaining complete oxidation while allowing reproducible measurements, with between day coefficients of variation of 2.35%, 14.6%, and 10.5% for lag, propagation and plateau phases, respectively. Beta-lipoproteins isolated from serum were more susceptible to oxidation than beta-lipoproteins from plasma apparently due to inhibition of oxidation by fibrinogen which co-eluted with beta-lipoprotein from plasma. For this reason, we recommend using serum preserved with EDTA for this assay.

Mechanized lipoprotein(a) assay as a marker for coronary artery disease illustrates the usefulness of high lipoprotein(a) levels.

Only a few simple lipoprotein(a) [Lp(a)] assays are available in kit form for use in clinical laboratories. The present study compares the analytical and clinical performance of a mechanized immunonephelometric method to enzyme-linked immunosorbent assay. Clinical performance was evaluated by measuring lipoprotein markers in 191 patients, with the extent of stenosis defined by angiography. Analytically, both methods showed little or no correlation with cholesterol, high density lipoprotein cholesterol, elevated triglycerides, apo A-I and apo B, while they showed good agreement with one another (r = 0.88). The methods showed comparable well known differences between black and white persons. Logistic regression indicated that Lp(a) was a weak but independent marker for coronary artery disease (CAD). Receiver operator characteristic curve analysis showed an association with CAD only at higher Lp(a) concentrations. We conclude that Lp(a) at higher concentrations may be a contributory marker for CAD and that mechanized nephelometric assays for it can be used in the clinical laboratory.

Studies of Bence Jones proteins by immunonephelometry.

Plasma cell dyscrasia is a disease caused by a monoclonal population of plasma cells. Most often the workup for this disease is prompted by the appearance of a paraprotein in serum, but a significant number of cases exhibit only a Bence Jones protein. Immunofixation electrophoresis is the most sensitive method presently used for identifying Bence Jones proteinuria. This methodology is laborious, expensive, and difficult to interpret. As a result, quantitative immunochemical methodologies have been suggested for measuring Bence Jones proteinuria. In this manuscript, it is demonstrated by rate nephelometry that: (1) although serum polyclonal immunoglobulins are accurately measured as compared to the calibration material, serum monoclonal immunoglobulins are not; (2) measurement of immunoglobulins in the urine of patients with generalized proteinuria is biased towards an increased number of light chains or a decreased number of heavy chains; and (3) Bence Jones proteins react with the assay antibodies differently from the calibration material and from one another. It is concluded that, although there is a qualitative relationship between concentrations of Bence Jones proteins and immunoglobulins used for calibration, measurement of absolute levels of Bence Jones proteins using currently available methods will lead to inaccuracies resulting from peculiarities between the antibody-antigen reaction and because of the spillover of polyclonal free light chains into urine. Nevertheless, the data provides credence for studies suggesting that measurement of the kappa/lambda ratio in serum and urine may be a reliable way to identify Bence Jones proteins by automated assays.

Urine protein electrophoresis and immunofixation electrophoresis supplement one another in characterizing proteinuria.

Urine protein electrophoresis (UPE) is often considered to have limited usefulness in evaluating proteinuria that is not associated with gammopathies. Unusual protein bands that are detected by UPE are commonly characterized by immunofixation electrophoresis (IFE). In this paper, electrophoretic gel patterns are shown to illustrate the greater sensitivity of IFE, compared to UPE. However, UPE remains useful for three applications: (1) UPE provides distinctive patterns that can indicate the source of proteinuria and is useful in assessing renal diseases that are independent of gammopathy; (2) combined use of UPE and IFE can avoid misinterpretations and repeated analyses of urine proteins, and (3) UPE can be used in conjunction with IFE to improve the quantitation of Bence-Jones proteinuria (BJP).

Problems with the measurement of apolipoproteins AI and AII.

There is considerable evidence demonstrating that increased levels of low density lipoprotein (LDL) cholesterol and decreased levels of high density lipoprotein (HDL) cholesterol are associated with coronary artery disease (CAD). Yet, these lipoprotein markers are insensitive for identifying risk of CAD. Measurement of subcomponents of HDL may offer more sensitive markers. Investigators have focused on protein components (apolipoproteins) of HDL as a potentially important marker. Unfortunately, because much of the immunoreactivity of apolipoproteins is hidden as a result of an association with lipids, it is difficult to measure them accurately. Detergents and other denaturing agents have been used to expose immunoreactivity. Poor correlations among different methods suggest that some detergents presently used may not be adequate for effective measurement of apolipoprotein (APO) AI. Studies using denaturing agents to probe HDL particles indicate that APO AII immunoreactivity is more resistant to exposure than that of APO AI. Data presented here indicate that the immunoreactivity of APO AII can be increased up to 50 percent by treatment with 4M guanidine provided the concentration of guanidine is diluted to less than 50 mM in the assay system. Previous studies failed to notice this effect because high levels of guanidine inhibited the antibody-antigen reaction in the immunoassay, making it appear that APO AII had not been exposed. It is concluded that with our present state of knowledge, it is unclear which, if any apolipoprotein assays, are adequately designed to achieve optimal exposure of antigenic sites, but that pretreatment with guanidine may be a simple, effective way to optimize APO AI and APO AII assays for clinical purposes.

Relationship between bilirubin, apolipoprotein B, and coronary artery disease.

Lipoprotein lipids and apo B from 254 male patients were compared with bilirubin as a risk factor for coronary artery disease (CAD). Patients were classified as: 1, Normal, all vessels < 20% stenosis, n = 83; or 2, CAD, at least one vessel > 70%, n = 171. Diagnostic accuracy was assessed by receiver operator characteristic curves (ROC). Corrections were made for possible confounding variables in the multivariate analysis. Upon ROC analysis, bilirubin showed an inverse relationship with risk of CAD, with areas under the ROC curve comparable to lipoprotein: however, bilirubin showed no discrimination below false positive frequencies of approximately 0.3. Logistic regression indicated that bilirubin was a weaker global marker than the lipoproteins and interacted with apo B. A highly significant correlation was found between bilirubin and apo B (p = 0.0025), but not with cholesterol, triglycerides, or high density lipoprotein cholesterol. Compared to lipoprotein markers, bilirubin provides little practical power of discrimination for CAD. Further studies of the affect of bilirubin on CAD must take its interaction with apo B into consideration.

Usefulness of various lactate dehydrogenase isoenzyme 1 profiles after myocardial infarction.

Isoenzyme profiles of lactate dehydrogenase (percent LD-1 of total LD, LD-1/LD-2 ratio, and absolute LD-1) have all been studied as late markers for myocardial infarction. It is known, however, that elevations of LD-5 frequently occur in this period as a result of liver congestion. Elevations of LD-5 may also occur as a result of complicating conditions. Such elevations could result in a reduced percent LD-1 of total LD, giving rise to false negatives. Receiver operating characteristic (ROC) curves were constructed for LD-1, LD-1/LD-2 and percent LD-1 of total LD from 285 specimens (124 patients) with suspected myocardial infarction. There was little difference in overall diagnostic power among the three assays. Using cutoffs determined from the ROC curves, 6 patients (18 specimens) were evaluated who appeared to be in the late period or who exhibited complicating conditions which could increase LD-5. In 14/18 specimens, increases in LD-5 resulted in false negatives by percent LD-1 of total LD. Only 5/18 specimens were false negatives by LD-1 or LD-1/LD-2. It is concluded that the percent LD-1 of total LD was affected by an increase in LD-5, and caution is recommended when using it.